CReasPy-Cloning: A Method for Simultaneous Cloning and Engineering of Megabase-Sized Genomes in Yeast Using the CRISPR-Cas9 System

Over the past decade,
a new strategy was developed to bypass the
difficulties to genetically engineer some microbial species by transferring
(or “cloning”) their genome into another organism that
is amenable to efficient genetic modifications and therefore acts
as a living workbench. As such, the yeast Saccharomyces cerevisiae has been used to clone and engineer genomes from viruses, bacteria,
and algae. The cloning step requires the insertion of yeast genetic
elements in the genome of interest, in order to drive its replication
and maintenance as an artificial chromosome in the host cell. Current
methods used to introduce these genetic elements are still unsatisfactory,
due either to their random nature (transposon) or the requirement
for unique restriction sites at specific positions (TAR cloning).
Here we describe the CReasPy-cloning, a new method that combines both
the ability of Cas9 to cleave DNA at a user-specified locus and the
yeast’s highly efficient homologous recombination to simultaneously
clone and engineer a bacterial chromosome in yeast. Using the 0.816
Mbp genome of Mycoplasma pneumoniae as a proof of
concept, we demonstrate that our method can be used to introduce the
yeast genetic element at any location in the bacterial chromosome
while simultaneously deleting various genes or group of genes. We
also show that CReasPy-cloning can be used to edit up to three independent
genomic loci at the same time with an efficiency high enough to warrant
the screening of a small (<50) number of clones, allowing for significantly
shortened genome engineering cycle times.